Epilepsy MERFISH Report

1. Overview

  • Project: Epilepsy MERFISH Analysis

1.1 Sample Information

A brief sample information is generated from the submission table for the following analysis.

Sample Index and Basic Information
Expt Case CHOC_Path_ID Sample_Index Sample_Region Age Genotype Region DataPath Comment
1 1300 SP24-1300-1A 1300_A7 A7 Junior Epilepsy region_0 Y:_Imaging_data_2\202409191416_20240919Ivy1300A7B12H500ADBCP1407x02_VMSC05201 Exclude because not enough cells contained
1 1300 SP24-1300-1B 1300_B11 B11 Junior Epilepsy region_1 Y:_Imaging_data_2\202409191416_20240919Ivy1300A7B12H500ADBCP1407x02_VMSC05201 NA
2 4_12112023 SP24-1463-1A 41211_TLE Temporal Lobe Junior Epilepsy region_0 Y:_Imaging_data_2\202409191416_20240919Ivy41211H500ADBCP1407x01_VMSC00101 NA
2 4_12112023 SP24-1463-1A 41211_TLE_dup Temporal Lobe Junior Epilepsy region_1 Y:_Imaging_data_2\202409191416_20240919Ivy41211H500ADBCP1407x01_VMSC00101 NA
3 NS02 NA NS02_TLE Temporal Lobe Adult Epilepsy region_0 Z:_Imaging_data_17_DATA_202202141032_20220214-NS02Ctx300VZG172_VMSC00101 NA
4 NS01 NA NS01_TLE Temporal Lobe Adult Epilepsy region_0 Z:_Imaging_data\202112261340_122621Lobotomy01-Ctx-frzn-300GPM_VMSC00101 NA

1.2 Sample Collection Form

Sample Collection with Original ID
CASE.# FFPE.ID Flash.Frozen.ID Region Immunohistochemistry.interpretation CHOC.Path.ID IRB.Net.ID Internal.Ref.# Xu.Lab.Received.FFPE
1 1-2B 2 parietal lobe Neu-N positive in neurons, highlights dysmorphic neurons and abnormal cortical lamination, including increased neurons in molecular layer; positive NFP for dysmorphic neurons; GFAP negative for ballon cells, highlights reactive astrocytes, Vimentin negative for balloon cells. SP23-2997-2 2059961-2 230456 A2
1 1-4B 4 temporal lobe Neu-N positive in neurons, demonstrates abnormal cortical lamnination (microcolumns); NFP positive in dysmorphic neurons; GFAP negative for balloon cells, highlights reactive astrocytes; Vimentin negative for balloon cells SP23-2997-4 2059961-2 230456 A3
1 1-4C n/a temporal lobe Neu-N positive in neurons, demonstrates abnormal cortical lamnination (microcolumns); GFAP highlights reactive astrocytes SP23-2997-4 2059961-2 230456 A4
1 1-5A 5 hippocampus Neu-N positive in neurons; GFAP highlights reactive astrocytes. SP23-2997-5 2059961-2 230456 A5
1 n/a 1 occiptial lobe n/a SP23-2997-1 2059961-2 230456 NA
1 n/a 3 frontal lobe n/a SP23-2997-3 2059961-2 230456 NA
2 2-1F 1 right frontal , epileptogenic focus grey and white matter with large foci showing marked gliosis, abundant hemosiderin deposition, axonal spheroids, and white matter pallor/tissue loss. SP23-4022 2059961-2 230456 1F
3 1A A contact 7 tissue at contact #7. Left frontal tuber SP24-1300-1A 2059961-2 230456 1A
3 1B B contact 12 tissue at contact #12. Left frontal tuber SP24-1300-1B 2059961-2 230456 1B
3 1C C random remainder of specimen. Left frontal tuber SP24-1300-1C 2059961-2 230456 1C
3 2A n/a NA Deep L frontal tuber / bottom of sulcus SP24-1300-2A 2059961-2 230456 2B
3 3A n/a NA Lateral tuber SP24-1300-3A 2059961-2 230456 3A
4 1A 1,2 left temporal (sample bag label:12112023, temporal lobe) SP24-1463-1A 2059961-2 230456 NA
4 2A 3,4 left frontal NA SP24-1463-2A 2059961-2 230456 NA
4 3B NA left lateral temporal NA SP24-1463-3B 2059961-2 230456 NA
4 4A NA amygdala NA SP24-1463-4A 2059961-2 230456 NA
4 5A NA hippocampus NA SP24-1463-5A 2059961-2 NA NA
5 NA NA NA NA NA NA NA NA

1.3 Sample Slice Pictures

NS01 Slice Experiment Note

NS01 Slice Experiment Note

NS02 Slice Experiment Note

NS02 Slice Experiment Note

2. Data Quality Control

2.1 MERSCOPE Quality Summary

The summaries present the data quality assessment automatically generated by MERSCOPE for each experiment. We mainly focus on the transcripts level for each sample. So we’re looking for high density in transcripts, based on the transcripts count per field of view (FOV), transcript density in FOV, and frequency of transcripts detected.

Generally, log10 transcript count > 4.0 in most area can be considered as a good quality standard.

Need to note that the low accuracy in DAPI cell boundary is not a concern, as a self-designed cell segmentation processing will take over this task.

2.1.1 (Bad Quality!) 1300_A7

2.1.2 1300_B11

2.1.3 41211_TLE

2.1.4 41211_TLE_dup

2.1.5 NS02_TLE

2.1.6 NS01_TLE

2.2 Bad Sample: Low Cell Count Captured

Cell Detection Comapre on Bad Sample

Cell Detection Comapre on Bad Sample

3. Data Processing & Analysis

3.1 Cell Segmentation & Filtering

Based on the spatial information and images obtained from MERFISH, we developed a machine learning model using the Cellpose algorithm to distinguish individual cells via MERFISH DAPI images.

To ensure the data quality and accuracy of cells, we have defined the minimum and maximum values for cell volume and gene count per cell. The cell volume should be between [100, 2500], and the gene count per cell > 25. After filter the outliers, the qualified cells count is shown in the following table.

Outliers were filtered from the data, and the qualified cell count is presented below. The transcript count Violin and transcript count Spatial Map are displayed here as part of the quality control reveal.

3.1.1 Cell Count after Filtering

Cell Total Count After Filtering
Sample Index Cell Count
X1300_B11 22938
X41211_TLE 36727
X41211_TLE_dup 31720
NS01_TLE 57074
NS02_TLE 51179

3.1.2 Transcript Count Violin

Transcript Count Violin After Filtering

Transcript Count Violin After Filtering

3.1.3 Transcript Count Spatial Map

Transcript Count Spatial Map After Filtering

Transcript Count Spatial Map After Filtering

3.2 Batch Effect & Dimension Reduction

We use Scanpy for the analysis of single-cell level transcriptome data. The initial stage of our analysis involves the elimination of batch effects, thereby ensuring that different samples from various batches are distributed within the same domain and are statistically reasonable to be integrated and compared. To achieve this, we utilize the Harmony algorithm.

Subsequently, we present visualizations of the batch difference by Leiden UMAP clusters. Also, we illustrate the distributions of the Leiden clusters for future analysis.

Umap of cells and colored by batch

Umap of cells and colored by batch

4. Cell Annotation

We use a recent published tool: Map My Cell to perform cell type annotations for each cell. It is a high resolution cell type annotator build by Alan Institude, with nested levels of classification including 34 classes and 338 subclasses.

The taxonomy is based on the Allen Mouse Brain Common Coordinate Framework version 3 (CCFv3)[https://doi.org/10.1016/j.cell.2020.04.007]. Part of the used abbreviations is list in the supplementary Abbreviation. Otherwise can be found in CCFv3 paper.

With the annotation, we can identify and plot the selected types of cells in each sample.

4.1 Cell Type Umap

Cells are annotated from low resolution to high resolution as follows:

4.2 Cell Type Spatial Map

4.3 Glutamatergic-only Spatial Map

4.4 Cell Type Count Table

Cell Type Count
cell_type_new X1300_B11 X41211_TLE X41211_TLE_dup NS01_TLE NS02_TLE Total
Astrocyte 3391 3915 3548 7519 5677 24050
Endothelial 1315 1909 1692 4553 2795 12264
Microglia 2871 3697 3135 5323 5581 20607
OPC 1392 1846 1642 3837 8666 17383
Oligo 7380 15486 13732 14265 12096 62959
VLMC 718 980 857 2663 2344 7562
L2/3/4 IT 979 2022 1703 5854 3509 14067
L5/6 IT 2235 3663 2790 6395 4336 19419
L5/6 NP 153 278 149 287 177 1044
L6b 846 573 517 507 387 2830
Lamp5 195 290 235 682 599 2001
Pvalb 402 552 466 1334 1092 3846
Sst 713 868 742 1717 1294 5334
Vip 348 648 512 2138 2626 6272
Total 22938 36727 31720 57074 51179 199638

5. Gene differentiation expresssion (to be done)

6. Cell communication via Cellchat (to be done)

Supplement: Abbreviation

Cell types & Regions

Astro, Astrocyte;

ABC, arachnoid barrier cells;

BAM, border-associated macrophages;

BLA, Basolateral amygdala;

CB, cerebellum;

CGE, caudal ganglionic eminence;

CHOR, choroid plexus;

CNU, cerebral nuclei;

CR, Cajal–Retzius;

CT, corticothalamic;

CTX, cerebral cortex;

CTXsp, cortical subplate;

DC, dendritic cells;

DCO, dorsal cochlear nucleus;

DG, dentate gyrus;

EA, extended amygdala;

Endo, endothelial cells;

ENT, Entorhinal area;

ENTl, Entorhinal area, lateral part;

Epen, ependymal;

EPI, epithalamus;

ET, extratelencephalic;

GC, granule cell;

HB, hindbrain;

HPF, hippocampal formation;

HY, hypothalamus;

HYa, anterior hypothalamic;

IMN, immature neurons;

IT, intratelencephalic;

L6b, layer 6b;

LGE, lateral ganglionic eminence;

LH, lateral habenula;

LSX, lateral septal complex;

MB, midbrain;

MGE, medial ganglionic eminence;

MH, medial habenula;

MM, medial mammillary nucleus;

MY, medulla;

NN, non-neuronal;

NP, near-projecting;

NT, non-telencephalon;

OB, olfactory bulb;

OEC, olfactory ensheathing cells;

OLF, olfactory areas;

Oligo, oligodendrocytes;

OPC, oligodendrocyte precursor cells;

P, pons;

PAL, pallidum;

Peri, pericytes;

PIR, piriform cortex;

SMC, smooth muscle cells;

STR, striatum;

TE, telencephalon;

TH, thalamus;

UBC, unipolar brush cells;

VLMC, vascular leptomeningeal cells.

Neurotransmitter types

Chol, cholinergic;

Dopa, dopaminergic;

GABA, GABAergic;

Glut, glutamatergic;

Glyc, glycinergic;

Hist, histaminergic;

Nora, noradrenergic;

Sero, serotonergic

ADP, anterodorsal preoptic nucleus

AHN, anterior hypothalamic nucleus

ARH, arcuate hypothalamic nucleus

CLI, central linear nucleus raphe

CUN, cuneiform nucleus

DMH, dorsomedial nucleus of the hypothalamus

DMX, dorsal motor nucleus of the vagus nerve

IF, interfascicular nucleus raphe

LHA, lateral hypothalamic area

MDRN, medullary reticular nucleus

MPN, medial preoptic nucleus

MPO, medial preoptic area

MV, medial vestibular nucleus

NTS, nucleus of the solitary tract

PAG, periaqueductal grey

PARN, parvicellular reticular nucleus

PB, parabrachial nucleus

PBG, parabigeminal nucleus

PGRN, paragigantocellular reticular nucleus

PGRNd, paragigantocellular reticular nucleus, dorsal part

PH, posterior hypothalamic nucleus

PMv, ventral premammillary nucleus

PPN, pedunculopontine nucleus

PVa, periventricular hypothalamic nucleus, anterior part

PVHd, paraventricular hypothalamic nucleus, descending division

PVi, periventricular hypothalamic nucleus, intermediate part

PVpo, periventricular hypothalamic nucleus, preoptic part

PVR, periventricular region

RAmb, midbrain raphe nuclei

RL, rostral linear nucleus raphe

SBPV, subparaventricular zone

SNc, substantia nigra, compact part

SPIV, spinal vestibular nucleus

TMv, tuberomammillary nucleus, ventral part

VII, facial motor nucleus

VMPO, ventromedial preoptic nucleus

VTA, ventral tegmental area

ZI, zona incerta.